Treatment of diabetes with copper binding compounds

ABSTRACT

Novel methods of treating a patient for diseases, disorders, and conditions including diabetes mellitus, comprising administering, for example, copper binding compounds.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/226,685, filed Aug. 23, 2002 (now U.S. Pat. No. 6,897,243), which isa continuation of U.S. Ser. No. 09/975,751, filed Oct. 10, 2001 (nowU.S. Pat. No. 6,610,693), which is a continuation application of U.S.Ser. No. 09/671,967, filed Sep. 27, 2000 (now U.S. Pat. No. 6,348,465),which is a National Stage application of international applicationPCT/NZ99/00161 (now published as WO/00/18392), filed Sep. 24, 1999,which claims the benefit of priority to New Zealand application 337042,filed Aug. 9, 1999; New Zealand application 334471, filed Mar. 3, 1999;New Zealand application 332079, filed Sep. 28, 1998; and New Zealandapplication 332084, filed Sep. 25, 1998; all documents listed herein arehereby incorporated by reference in their entirety.

FIELD

The present invention is in the field of biochemistry. Morespecifically, the invention involves fructosamine oxidase enzymeinhibitors. Methods of treatment, pharmaceutical compositions, dosageforms, uses of fructosamine oxidase enzyme inhibitors in medicine or formanufacturing pharmaceutical compositions, treatment regimes, andrelated combinations, methods and products are disclosed herein.

BACKGROUND

Diabetes mellitus is a common disorder affecting nearly 16 millionAmericans. See, for example, Report of the Expert Committee on theDiagnosis and Classification of Diabetes Mellitus. Diabetes Care, 20;1183-97 (1997). Diabetic individuals are prone to complications whichare a major threat to both the quality and the quantity of life. Almosthalf those diagnosed with diabetes before the age of 31 years die beforethey reach 50 years largely as a result of cardiovascular or renalcomplications, often with many years of crippling and debilitatingdisease beforehand. See, Deckert T., Poulsen J., Larsen M. Diabetologia14: 363-70 (1978). It is estimated that diabetic individuals have a25-fold increase in the risk of blindness, a 20-fold increase in therisk of renal failure, a 20-fold increase in the risk of amputation as aresult of gangrene, and a 2- to 6-fold increased risk of coronary heartdisease and ischemic brain damage. See, Klein R., Klein B., Moss S.,Davis M., DeMets D. Diabetes Care 8; 311-5 (1985).

Largely because of these long-term complications, the cost of diabetesin the US was estimated as $98 billion in 1997 comprising $44 billionfor direct medical costs such as inpatient and outpatient care plus $54billion for indirect costs such as lost earnings and productivity, andpremature death. Medical innovations that can slow the progression ofdiabetes have tremendous potential to mitigate the associated clinicaland cost repercussions. See, American Diabetes Association, “Economicconsequences of diabetes in the US in 1997,” Diabetes Care 21: 296-309(1998).

Elevated blood glucose levels are now regarded as causative of diabeticcomplications based on results of the Diabetes Complications and ControlTrial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS).See, N Eng J. Med. 379: 977-85 (1993) and Lancet 352: 837-53 (1998). TheDCCT and the UKPDS have both demonstrated that the development ofcomplications of diabetes is related with the degree of hyperglycemiaand that the long-term outcome may be ameliorated by rigorous treatment.For example, prognosis is dramatically improved if capillary blood andglycated hemoglobin levels are maintained less than 150 mg/dL and 7.0%respectively.

The mechanism of glucose toxicity in the tissues of patients withdiabetes mellitus is unknown. Glucose condenses with free amino groupson structural and functional proteins to form Schiff bases which, inturn, undergo a series of transformations to yield dark-brown Maillardproducts. It has been proposed that diabetes complications are caused bythe non-enzymatic cross-linking of proteins. See, for example, CeramiA., Ulrich P. C., Brownlee M., U.S. Pat. No. 4,758,583 (1988). However,although increased protein cross-linking is seen in the tissues ofpeople with long-standing diabetes, the role of Maillard products as acausative factor is certainly not clear. See, for example, Wolff S. P.,Jiang Z. Y., Hunt J. V. Free Rad Biol Med 10; 339-52 (1991).

Amadori-rearrangement is the most important Maillard transformationbecause its product, fructosamine, is the precursor of all the browningproducts. A novel extracellular enzyme which catalyzes the eliminationof fructosamines from glycated protein has been isolated. Enzymes whichare related have been disclosed. See, for example, Gerhardinger C., etal. J Biol Chem 270 (1): 218-24 (1995); Saxena, A. K. et al., J BiolChem 271 (51): 32803-9 (1996); and Horiuchi T, et al., Agric. Biol.Chem. 53 (1): 103-110 (1989). Based on its high specificity for glycatedprotein substrates and its use of oxygen as an acceptor, the enzyme maybe classified as fructosamine oxidase 1.5.3. See, Enzyme Nomenclature,Recommendations of the Nomenclature Committee of the International Unionof Biochemistry, Academic Press, London pp. 19-22, (1979).

Fructosamine oxidase is a copper metalloenzyme which belongs to thecopper amine oxidase group of enzymes which have previously beenisolated from bacteria, fungi, yeast, and mammalian sera. Products ofthe fructosamine oxidase catalyzed reaction are free unglycated protein,α-dicarbonyl sugar, and the active oxygen species superoxide. A highlyspecific copper chelator, triethylenetetramine dihydrochloride, is anirreversible inhibitor of fructosamine oxidase activity. See, forexample, Morpurgo L, et al. Biol Met 3: 114-7 (1990).

Increased fructosamine oxidase activity may cause many of the recognizedsequelae of diabetes by degrading fructosamines bound to basementmembrane proteins and generating reactive oxygen species as reactionproducts. For example, superoxide anions cause an increase inintracellular calcium which modulates the activity of nitric oxidesynthase. Nitric oxide is a potent vasodilator and it has beenimplicated in the vascular dysfunction of early diabetes. See, forexample, Ido Y., Kilo C., Williamson J. R. Nephrol Dial Transplant 11Suppl 5: 72-5 (1996). Reactive oxygen species also cause a drasticdose-dependent decrease in de novo synthesis of heparin sulfateproteoglycans leading to a reduction in anionic sites on the glomerularbasement membrane and an increase in basement membrane permeability tocationic plasma proteins such as albumin. See, Kashira N., Watanabe Y.,Malin H., Wallner E. I., and Kanwar Y. S. Proc Natl Acad Sci USA 89:6309-13 (1992). Increased urinary albumin clearance is a risk indicatorin people with diabetes mellitus both for evolving renal disease and forearly mortality mainly from coronary heart disease. See, for example,Mattock M. B., Barnes D. J., Viberti G. C., et al. Diabetes 47: 1786-92(1998).

Once natural anti-oxidant defenses are exceeded, hydroxyl radicals maybe generated from superoxide via a copper catalyzed Haber-Weissreaction. See, Halliwell B. and Gutteridge J. M. C. “Free radicals inBiology and Medicine” Clarendon Press, Oxford pp. 136-76 (1989).Hydroxyl radicals are extremely reactive species and could cause thepermanent site-specific damage to basement membrane proteins andhistopathological changes that are typical of diabetic microvasculardisease. See, Robbins S. L., Cotran R. S., Kumar V. “Pathologic basis ofdisease” 3^(rd) ed. W. B. Saunders, pp. 991-1061. (1984).

Similarly, any prolonged increase in fructosamine oxidase activity willcause oxidative stress which could account for the excess risk ofmacrovascular disease and the 75% increase in mortality seen in patientswith diabetes mellitus compared with non-diabetic individuals. Recentstudies have convincingly demonstrated that oxidative modification oflow density lipoprotein (LDL) is involved in the development ofatherosclerosis of coronary and peripheral arterial vessels and elevatedoxidized LDL concentrations are found in subjects with diabetesmellitus. See, Witztum J. L. Br Heart J 69 (Suppl): S12-S18 (1993) andPicard S., Talussot C., Serusclat A. et al. Diabetes and Metabolism 22:25-30 (1996). Oxidative changes to membrane lipids and to membraneprotein SH-groups may also cause aberrations in cellular calciumhomeostasis and contribute to the increased incidence of cardiac suddendeath that is typical of diabetes. See, Yucel D., Aydogdu S., Cehreli S.et al. Clin Chem 44: 148-54 (1998).

Triethylenetetramine dihydrochloride, also known as trienes ortrien-2HCl or trientine dihydrochloride, is a copper chelating agent.Trienes have been used for treating individuals with Wilson's disease.See, for example, Dubois R. S., Lancet 2 (7676): 775 (1970); Walsh, J.M., Q J. Med. 42 (167): 441-52 (1973); Haslam, R. H., et al., DevPharmacol Ther 1 (5): 318-24 (1980). Trienes have also been used totreat individuals with primary biliary cirrhosis. See, for example,Epstein O., et al., Gastroenterology 78 (6): 1442-5 (1980). In addition,trienes have been used to inhibit the spontaneous development ofhepatitis and hepatic tumors in rats. See, for example, Sone K., et al.,Hepatology 23 (4): 764-70 (1996). Thus far, trienes have not been usedin the treatment of diabetes.

All publications and patents cited herein are hereby incorporated byreference in their entirety.

BRIEF SUMMARY

Excess fructosamine oxidase activity with glycated basement membraneprotein substrate plays a vital role in diabetic complications by theformation of α-dicarbonyl and reactive oxygen free radical species.

This damage may be ameliorated by administering specific fructosamineoxidase inhibitors or antagonists selected from the groups: (i) copperchelating agents; (ii) substrate analogues; and (iii) hydrazinecompounds.

In one aspect, the present invention consists in a method of treating anindividual (human or otherwise) predisposed to and/or suffering fromdiabetes mellitus with a view to minimizing the consequences ofmacrovascular and microvascular damage to the patient (e.g., acceleratedatherosclerosis, blindness, renal failure, neuropathy, etc.) whichcomprises, in addition to any treatment in order to control bloodglucose levels, at least periodically inhibiting or antagonizingfructosamine oxidase enzyme activity in the patient.

Preferably said inhibition, or antagonism occurs as a result ofadministration or self-administration of at least one fructosamineoxidase reaction product inhibitor or antagonist.

Preferably any such inhibitor or antagonist is selected from the groups:

(i) copper chelating agents;

(ii) substrate analogue; and/or

(iii) hydrazine compound.

Preferably said inhibitor or antagonist is taken orally.

Preferably said inhibitor or antagonist is taken orally as part of aregime, whether totally oral or not, which also involves the control ofblood glucose levels.

In a further aspect, the present invention consists in a pharmaceuticalcomposition (preferably oral) suitable for use in such a method, saidcomposition comprising a fructosamine oxidase inhibitor or antagonist inconjunction with a suitable carrier therefor.

In yet a further aspect, the present invention consists in apharmaceutical composition for reducing macrovascular and microvasculardamage in an individual (including a human) suffering from diabetesmellitus, said composition comprising a fructosamine oxidase inhibitoror antagonist and a suitable carrier therefor.

Preferably said carrier can be any diluent, excipient or the like andthe dosage form of said pharmaceutical composition can be of anyappropriate type whether for oral or other administration orself-administration. Long acting release forms are also envisaged withinthe present invention.

In still a further aspect, the present invention consists in the use ofa fructosamine oxidase inhibitor or antagonist in the manufacture of apharmaceutical composition comprising the fructosamine oxidase inhibitoror antagonist and a suitable pharmaceutical carrier therefor and whichcomposition is useful in treating an individual (human or otherwise)which or who is suffering from diabetes mellitus to reduce macrovascularand microvascular damage (preferably by a method of the presentinvention).

In still a further aspect, the present invention consists incombination, the treatment regimes, and/or the medicaments of suchregimes previously set forth whether packed together or prescribedtogether or otherwise.

In still another aspect, the invention consists in a method of treatingan individual (human or otherwise) predisposed to and/or suffering fromdiabetes mellitus, which includes inhibiting or antagonizingfructosamine oxidase enzyme activity in the patient with an agent oragents preferably not contraindicated for the patient. Examples ofinhibitors or antagonists include but are not limited to those listedhereinafter.

Preferably in one embodiment said agent(s) is or are copper chelatingcompound(s) administered or self-administered to the patient.

Examples of suitable copper-chelating compounds includetriethylenetetramine dihydrochloride, penicillamine, sar, diamsar,ethylenediamine tetraacetic acid, o-phenanthroline, and histidine.

Preferably in another embodiment, said agent(s) is or are substrateanalogue compound(s) administered or self-administered to the patienthaving an amino acid or peptide moiety with a blocked N-terminal aminegroup.

Examples of a suitable substrate analogue composition areN-acetylcysteine, captopril, lisinopril and enalapril.

Preferably in another embodiment said agent(s) is or are hydrazinecompound(s) administered or self-administered to the patient i.e., acompound having a —NHNH₂ moiety.

Examples of a suitable hydrazine compound include diaminoguanidine,hydralazine, and carbidopa.

In still another aspect, the invention consists in a dosage regimen fora method of the present invention and/or using dosage units of thepresent invention.

In still a further aspect, the present invention consists in the use ofpharmaceutically acceptable compounds being at least one of a substrateanalogue, a hydrazine compound and a copper chelator in the manufactureof a dosage unit or pharmaceutical composition useful in treating anindividual (human or otherwise) which or who is suffering from diabetesmellitus to reduce macrovascular and microvascular damage.

In another aspect, the invention consists in a dosage unit orpharmaceutical composition for an individual useful in a method of thepresent invention comprising (preferably in effective fructosamineoxidase reaction product-inhibiting or -antagonizing amounts -separatelyor collectively) of a compound (or compounds) being a substrate analogueor a hydrazine compound having an —NHNH₂ moiety, or both.

Preferably said dosage unit also includes said pharmaceuticalcomposition that also includes one or more compounds which are copperchelators.

Preferably said dosage unit or composition is in an oral dosage formoptionally with carriers, excipients or, indeed, even other activeagents (e.g., means to lower blood glucose levels).

In still another aspect, the invention consists in a regime or dosageunit or pharmaceutical composition for a diabetic or suspected diabeticindividual of the copper chelator, triethylenetetramine dihydrochloride,providing for the patient a sufficient fructosamine oxidase inhibitingand/or antagonizing effect to reduce macrovascular and microvasculardamage.

In still another aspect, the invention consists in a regime or dosageunit or pharmaceutical composition of captopril for a diabetic orsuspected diabetic individual, whether effective or intended to beeffective in controlling the blood pressure of the diabetic patient (atleast in part) or not, providing for the patient a sufficientfructosamine oxidase inhibiting and/or antagonizing effect to reducemacrovascular and microvascular damage.

In yet another aspect, the invention consists in a regime or dosage unitor pharmaceutical composition for a diabetic patient or suspecteddiabetic patient of a hydrazine compound providing for the patient asufficient fructosamine oxidase inhibiting and/or antagonizing effect toreduce macrovascular and microvascular damage.

In yet another aspect, the invention consists in a regime or dosage unitor pharmaceutical composition for a diabetic patient or suspecteddiabetic patient of

(i) acetylcysteine and

(ii) at least one other fructosamine oxidase inhibitor and/orantagonist, the mix of (i) and (ii) providing for the patient asufficient fructosamine oxidase inhibiting and/or antagonizing effect toreduce macrovascular and microvascular damage.

In yet another aspect, the invention consists in a regime or dosage unitor pharmaceutical composition for a diabetic patient or suspecteddiabetic patient of

(i) hydralazine and

(ii) at least one other fructosamine oxidase inhibitor and/orantagonist, the mix of (i) and (ii) providing for the patient asufficient fructosamine oxidase inhibiting and/or antagonizing effect toreduce macrovascular and microvascular damage.

In still another aspect, the present invention consists in a method oftreating an individual (human or otherwise) predisposed to and/orsuffering from diabetes mellitus which includes inhibiting and/orantagonizing fructosamine oxidase enzyme activity in the patient withacetylcysteine and hydralazine.

In still another aspect, the invention consists in a regime or dosageunit or pharmaceutical composition for a diabetic or suspected diabeticindividual which includes acetylcysteine and hydralazine.

In still a further aspect, the present invention consists in the use ofor co-administration or serial administration of acetylcysteine andhydralazine for the purpose of reducing macrovascular and microvasculardamage in an individual.

Preferably said individual is diabetic.

In yet another aspect, the invention consists in a method of treatingand/or reducing the likelihood of diabetic cataract formation in anindividual which comprises at least periodically inhibiting and/orantagonizing fructosamine oxidase enzyme activity in the mammal.

Preferably the method involves the administration or self-administrationof effective amounts of triethylenetetramine dihydrochloride (or othertriene).

In another aspect, the invention consists in a method of treating and/orreducing the likelihood of diabetic cardiomyopathy in an individualwhich comprises at least periodically inhibiting and/or antagonizingfructosamine oxidase enzyme activity in the individual.

Preferably the method involves the administration or self-administrationof effective amounts of triethylenetetramine dihydrochloride (or othertriene).

Preferably for any of the aforesaid indications triethylenetetraminedihydrochloride (or other triene) is administered and/or selfadministered in concert with another (other) fructosamine oxidase enzymeinhibitor(s) and/or antagonist(s).

Preferably said another inhibitor and/or antagonist or said otherinhibitors and/or antagonists is or are administered or selfadministered to elicit a pharmacological effect for another indicationyet together with the effect of the triene is or are in an amount oramounts which are effective for treating or ameliorating macrovascularand microvascular damage of such an individual.

Reference is drawn to PCT Application PCT/NZ99/00161, filed Sep. 24,1999 (claiming priority of New Zealand Patent Specification No. 332085filed Sep. 25, 1998), the full content of which is hereby incorporatedby reference. It discloses methods of monitoring fructosamine oxidaseinhibition and/or antagonism of patients, screening patients todetermine patients at risk to vascular (particularly microvascular)damage and identifying those individuals who will benefit by treatmentwith fructosamine oxidase inhibitors and/or antagonists, methods ofdetermining fructosamine oxidase levels in a mammal, methods ofdetermining blood plasma fructosamine oxidase levels in a diabeticindividual or a suspected individual, methods of assaying blood serum orblood plasma in vitro for fructosamine oxidase, methods of identifyingor testing candidate substances and to related methods and procedures.

Preferably the measurement conducted in vitro is of the superoxidereaction product (or any other oxygen free radical product) offructosamine oxidase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a detailed reaction mechanism for the formation offructosamine and Maillard products from glucose and protein.Fructosamine oxidase degrades fructosamine by a two-step reaction withinitial release of an α-dicarbonyl sugar and subsequent oxidation of theenzyme/protein complex to release free unglycated protein. The reducedcopper cofactor is oxidized in vivo by molecular oxygen and theoxidation product is superoxide.

FIG. 2 shows the absorbance spectra of the fructosamine oxidase enzymesextracted from pooled human sera (A) and from the microbial organism,Enterbacter aerogenes (B).

FIG. 3 shows the spectra of p-nitrophenylhydrazine (NPH) adduct of theEnterbacter aerogenes enzyme (A) and a red absorbance shift when theNPH-enzyme adduct is diluted in 2M KOH.

FIG. 4 shows the survival curve for non-treated STZ-diabetic ratscompared with diabetic animals treated with fructosamine oxidaseinhibitors.

FIG. 5 shows the monthly growth of treated and untreated STZ-diabeticrats compared with non-diabetic animals.

DETAILED DESCRIPTION

The present invention discloses the use of fructosamine oxidaseinhibitors to treat an individual with diabetes mellitus. In one aspect,the invention provides methods of treating diabetes by reducingfructosamine oxidase activity within an individual who is suffering fromdiabetes.

Definitions

As used herein (including in the claims), the term “inhibitor” is usedinterchangeably with “antagonist” and refers to a compound whichsubstantially reduces fructosamine oxidase activity.

The term “substantially reduces” refers to a reduction of fructosamineoxidase activity by about 5%, more preferably about 10%, even morepreferably about 20%, even more preferably about 30%, even morepreferably about 40%, even more preferably about 50%, even morepreferably about 60%, even more preferably about 70%, even morepreferably about 80%, even more preferably about 90%, even morepreferably about 100%.

The term “copper chelating agents” means any agent which reduces bodyfructosamine oxidase activity by lessening the availability of bodycopper stores and/or by the binding of said copper chelating agent tofructosamine oxidase enzyme. Such binding can have various effects, forexample, inactivation of the copper molecule at the reactive center ofthe enzyme, conformational changes that may affect the activity of theenzyme, or binding a non-reactive portion of the enzyme in a manner thataffects the activity of the enzyme. Binding can be either reversible orirreversible.

The term “substrate analogue” refers to any chemically modified aminoacid or peptide substrate which lessens the activity of fructosamineoxidase enzyme. A non-limiting example by which a substrate analogue canlessen the activity of fructosamine oxidase is by binding to the enzyme.Such binding can have various effects, for example, inactivation of thereactive center of the enzyme, conformational changes that may affectthe activity of the enzyme, or binding a non-reactive portion of theenzyme in a manner that affects the activity of the enzyme. Binding canbe either reversible or irreversible.

The term “hydrazine compound” means any agent containing the moiety—NH—NH₂ which lessens the activity of fructosamine oxidase enzyme. Anon-limiting example by which a hydrazine compound can lessen theactivity of fructosamine oxidase is by binding to the enzyme. Suchbinding can have various effects, for example, inactivation of thereactive center of the enzyme, conformational changes that may affectthe activity of the enzyme, or binding a non-reactive portion of theenzyme in a manner that affects the activity of the enzyme. Binding canbe either reversible or irreversible.

The term “at least periodically” includes from a single administrationto continuous administration.

The term “macrovascular damage” refers to accelerated atherosclerosis oflarge arteries supplying blood to the heart, to the lower limbs, and tothe brain. Macrovascular damage can be assessed by various organ imagingtechniques such as catheter/dye studies (angiograms) and magneticresonance angiography. Furthermore, in animal models, whole bodyresponses include, but not limited to, survival and/or weight gainand/or histopathological changes of cardiomyopathy can also be used toassess macrovascular damage. In humans, macrovascular damage can beassessed by any of the measured above as well as monitoring significantphysiological changes seen during clinical trial of differentfructosamine oxidase inhibitors.

The term “microvascular damage” refers to damage to small arterioles andcapillaries, for example, the arterioles and capillaries supplying bloodto the retina in the eye, the glomerulus in the kidney, and theperipheral and autonomic nervous system. Microvascular damage of theretina can be assessed by direct ophthalmoscopy, by slit lampmicroscopy, or by fluorescein angiography. Microvascular damageelsewhere may be assessed by surrogate measurements. For example,microvascular damage to the kidney glomerulus or peripheral nerves canbe assessed by its effect on tissue function, e.g., proteinuria reflectsdamage to the kidney glomerulus or nerve conduction studies reflectdamage to peripheral nerves.

“In concert with” does not necessarily mean as a result of simultaneousadministration or self-administration. It can be administered seriallyand such serial application can be spaced, i.e., a triene between mealsand another agent with a meal.

The terms “triethylenetetramine dihydrochloride” and “triene” are usedinterchangeably throughout and includes any pharmaceutically acceptablefructosamine oxidase enzyme inhibiting and/or antagonizing analogue ormetabolite thereof (e.g., an acetylated derivative) for the targetmammalian species or for a human being capable of administration or selfadministration in an amount alone or in concert with anotherfructosamine oxidase enzyme inhibitor and/or antagonist (preferably notcontraindicated by toxicity concerns having regard to levels requiredfor effective inhibition and/or antagonism), of providing effectiveinhibition and/or antagonism.

The term “an effective amount” of a fructosamine oxidase inhibitorrefers to the amount of one or more fructosamine oxidase inhibitorsrequired to ameliorate the physiological well-being of an individualsuffering from diabetes mellitus. This can involve the amount having aneffect on fructosamine oxidase activity in an individual to which theinhibitor(s) is being administered.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, humans and non-rodentpets.

“Comprises” can mean “includes”.

“And/or” means both “and” and “or”.

Use of Fructosamine Oxidase Inhibitors

Fructosamine oxidase inhibitors can be selected from any number ofcompounds from several different groups such as copper chelators,substrate analogs, and hydrazine compounds. These compounds are readilyavailable from commercial sources, for example, Sigma Chemical Company(St Louis, Mo.) or Aldrich Chemicals Milwaukee, Wis.). Preferably thecompound is a triene. The triene can be administered by any appropriateadministration route. Non-limiting examples include oral intake (i.e.,ingestion by eating or drinking), injection, and mucosal. In oneembodiment, a triene is administered in a pharmaceutically acceptablecomposition. In another embodiment, a triene is used as apharmaceutically acceptable composition in combination with anothercompound to lessen fructosamine oxidase activity. Preferably thecombination of the triene and a second compound act in cooperation witheach other to lower fructosamine oxidase activity. In yet anotherembodiment, a combination of at least two fructosamine oxidaseinhibitors are used in a pharmaceutically acceptable composition.Preferably, the inhibitors do not lessen the efficacy of each other whenadministered in combination. Preferably, a triene is administered with apharmaceutically acceptable excipient. A pharmaceutically acceptableexcipient is a relatively inert substance that facilitatesadministration of a pharmacologically effective substance. For example,an excipient can give form or consistency to the vaccine composition, oract as a diluent. Suitable excipients include but, are not limited tostabilizing agents, wetting and emulsifying agents, salts for varyingosmolarity, encapsulating agents, buffers, and skin penetrationenhancers. Examples of pharmaceutically acceptable excipients aredescribed in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro,ed., 18th edition, (1990). The dosage used is an effective amount of afructosamine oxidase inhibitor to substantially reduce fructosamineoxidase activity. Preferably, the dosage used is an effective amount ofa triene to substantially reduce fructosamine oxidase activity. In analternate embodiment, the dosage used is an effective amount tosubstantially reduce the symptoms of diabetes and its sequelae (renaldysfunction, visual dysfunction, cardiovascular disease, wound healingproblems, etc.). The dosage is preferably about 1 mg/kg to about 1 g/kg,more preferably about 2 mg/kg to about 800 mg/kg, even more preferablyabout 5 mg/kg to about 600 mg/kg, even more preferably about 7 mg/kg toabout 400 mg/kg, even more preferably about 9 mg/kg to about 200 mg/kg,even more preferably about 11 mg/kg to about 100 mg/kg, even morepreferably about 13 mg/kg to about 75 mg/kg, even more preferably about15 mg/kg to about 50 mg/kg, even more preferably about 17 mg/kg to about35 mg/kg. The administration can be as often as needed to achieve areduction in fructosamine oxidase activity. A skilled artisan maydetermine if the combination lessens the efficacy by a stepwiseadministration of a combination of fructosamine oxidase inhibitors atvarious dosages and measuring parameters exemplified in the Examplessuch as weight loss, etc. Fructosamine oxidase inhibitors, preferably atriene alone or in combination with other inhibitors, can beadministered to individual suffering from diabetes mellitus and itssequelae and/or to individuals who are susceptible to diabetes mellitus(i.e., genetic pre-disposition). Genetic pre-disposition can bedetermined by examination and analysis of family history of diabetesmellitus or by genetic markers correlated with development of diabetesmellitus.

EXAMPLES Example 1 Extraction of Holoenzyme

Fructosamine oxidase in blood plasma is largely found as anenzyme-substrate conjugate, bound to peptides and proteins (FIG. 1). Toobtain a maximal yield of active holoenzyme, it was necessary to makethe pH of the media alkaline preferably with phosphate buffer, to addsulphydryl reagents, and to incubate the mixture with pro-oxidant sothat glycated species were released. Most effective activation was foundwith cupric salts.

Fructosamine oxidase holoenzyme was separated from inactive apoenzyme byaffinity adsorption chromatography. A suitable glycated affinity supportwas prepared from alkylamine beads or beaded cross-linked agarose withamino terminal residues attached by 6-10 atom spacer arms (availablefrom Pierce™, Bio-Rad™, and Pharmacia™). The affinity support wasglycated by incubating with 400 mM potassium phosphate buffer pH 7.4containing 50 mM glucose and 0.01% sodium azide at 37° C. for 7 days.Holoenzyme bound tightly to glycated amino residues and residual copperwas readily removed by washing with water. Active holoenzyme was elutedwith 800 mM NaCl in 50 mM sodium acetate buffer pH 4.8. Active fractionswere pooled and protein was precipitated with 50% cold acetone solvent.The protein pellet was reconstituted with a minimum volume of water orphysiological saline and lyophilized for long term storage.

Fructosamine oxidase holoenzyme was separated from inactive apoenzyme byaffinity adsorption chromatography. A suitable glycated affinity supportwas prepared from alkylamine beads or beaded cross-linked agarose withamino terminal residues attached by 6-10 atom spacer arms (availablefrom Pierce™, Bio-Rad™, and Pharmacia™). The affinity support wasglycated by incubating with 400 mM potassium phosphate buffer pH 7.4containing 50 mM glucose and 0.01% sodium azide at 37° C. for 7 days.Holoenzyme bound tightly to glycated amino residues and residual copperwas readily removed by washing with water. Active holoenzyme was elutedwith 800 mM NaCl in 50 mM sodium acetate buffer pH 4.8. Active fractionswere pooled and protein was precipitated with 50% cold acetone solvent.The protein pellet was reconstituted with a minimal volume of water orphysiological saline and lyophilized for long term storage.

Extraction of 35 mL pooled diabetic and non-diabetic human sera yieldeda clear colorless preparation with absorbance peaks at 196 nm and 264 nmtypical of the absorbance spectra of fructosamine oxidase (FIG. 2). Afructosamine oxidase enzyme from Enterobacter aerogenes showingabsorbance peaks at 196 nm and 255 nm was included for comparison.Enzyme activity and relative activity was as follows.

TABLE I Protein Cytochrome c Sp activity† Sample (μg/mL) activity* (U/L)(U/g) human 32.9 4.58 139.4 E. aerogenes 541.5 66.32 115.11 *Enzymeextract was preincubated in 0.05 M TES buffer pH 7.4 containing 1 mM DMFsubstrate at 37° C. for 5 minutes. Enzyme activity was measured with 10μM ferricytochrome c. The reaction was started with 50 μM fructosaminesubstrate as g-BSA and ΔA_(550 nm) was determined over 5 minutes.†Protein concentration determined from A_(210 nm)-A_(220 nm) comparedwith BSA standards.

Cofactor Identification

The p-nitrophenylhydrazine (NPH) adduct of Enterbacter aerogenes enzymewith A_(max) 399 nm was obtained as described previously. See, Palcic M.M., Janes S. M. Meth Enzymol 258: 34-8 (1995). A red absorbance shift toA_(max) 438 nm was observed when the NPH-enzyme adduct was diluted in 2MKOH. Such an absorbance shift was typical of the quinone cofactors ofcopper amine oxidase.

Example 2 Identifying Fructosamine Oxidase Inhibitors

The purpose of this example was to demonstrate how the fructosamineoxidase assay, the subject of a PCT International patent specificationNZ 332085 the contents of which are hereby incorporated by reference,may be used in identifying and grading candidate fructosamine oxidaseinhibitors. This approach took into account the activity of the drug ina human plasma matrix in vitro. Enzyme inhibitors have wide and numerousapplications in clinical medicine as treatments for a range of metabolicdisorders. For example, angiotensin converting enzyme inhibitors havebeen used in the treatment of hypertension. See, for example, Harris E.E., Patchett A. A., Tristram E. W., and Wyvratt M. J., “Aminoacidderivatives as antihypertensives” U.S. Pat. No. 4,374,829 (1983).Similarly, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductaseenzyme inhibitors have been used in the treatment ofhypercholesterolemia. See, for example, Hoffman W. F., Smith R. L., LeeT. J., “Novel HMG-CoA reductase inhibitors” U.S. Pat. No. 4,866,090(1989). Fructosamine oxidase inhibitors may be selected from thosesubstances which bind and block the quinone co-factor (hydrazinecompounds), the copper co-factor (copper chelators), or which mimic thenormal substrate of the enzyme (substrate analogue).

Method:

Potential fructosamine oxidase inhibitors were tested on human serum orplasma individually and in combination) using the method of assayingfructosamine oxidase activity described in detail in a New ZealandPatent Specification No. 332085. Irreversible enzyme inhibition wascharacterized by a progressive decrease in activity with time ultimatelyreaching complete inhibition even with very dilute inhibitorconcentrations provided that the inhibitor is in excess of the amount ofenzyme present.

Results:

The relative activity of a selection of hydrazine, copper chelator, andsubstrate analogue Fructosamine oxidase inhibitors are shown in Table 2.In some instances, there was a degree of overlap between classes, i.e.,some hydrazine compounds were also copper chelators. To clarify thispoint, the copper chelating potential for some compounds is indicated(β). The effectiveness of the inhibitor was expressed not by anequilibrium constant but by a velocity constant (K) which determined thefraction of the enzyme inhibited in a given period of time by a certainconcentration of inhibitor. The specificity of the inhibitor for theactive center of the enzyme was indicated by the concentration ofinhibitor causing 50% inactivation of the enzyme (IC₅₀).

TABLE 2 IC₅₀ ¹ K(min⁻¹) β³ Inhibitor: Hydrazine compounds aminoguanidine231 μM 0.0067* — semicarbazide 45 μM 0.0276* +++ benserazide 13.6 μM0.0095* oxalic dyhydrazide 1.59 μM 0.0542 — hydralazine 1.52 μM 0.0029+++ phenylhydrazine 0.81 μM 0.1160 — carbidopa 0.50 μM 0.1496diaminoguanidine 0.36 μM 0.1340 — Inhibitor: Substrate analogueslisinopril 216.9 μM 0.0174 +++ enalapril 3.95 μM 0.0326 — captopril 1.78μM 0.0259 acetylpenicillamine 1.06 μM 0.0811 acetylcysteine 0.83 μM0.1677 Inhibitor: Copper chelators desferrioxamine 40.6 μM 0.0109* EDTA15.7 μM 0.0755* Sodium azide 9.48 μM 0.0004 Potassium cyanide 6.36 μM0.0116 triethylenetetramine dihydrochloride 5.40 μM 0.0196o-phenathroline 4.25 μM 0.0385 histidine 2.29 μM 0.0554 Inhibitor:Combined agents acetylcysteine + hydralazine 0.57 μM 0.1654acetylcysteine + diamino guanidine 1.07 μM 0.0795 acetylcysteine +histidine 1.11 μM 0.0722 acetylcysteine + carbidopa 0.27 μM 0.2000¹fresh human sera was incubated with 0-1,000 μM inhibitor in 0.05 M TESbuffer pH 7.4 at 37° C. for 5 minutes. Enzyme activity was measured with10 μM ferricytochrome c. The reaction was started with 50 μMfructosamine substrate as g-BSA and ΔA_(550 nm) was determined over 5minutes ²rate constants were calculated from the reaction offructosamine oxidase either with 1.0 μM inhibitor or with 10.0 μMinhibitor (*) ³the copper chelating potential (β) was determined fromthe ability of the agent to remove copper under dialysis fromcopper-saturated BSA substrate.

Conclusions:

Irreversible inhibition of fructosamine oxidase is feasible.

Inhibitors may be broadly categorized in three classes of compound:hydrazines; substrate analogues; and copper chelators.

Fructosamine oxidase activity in human blood plasma may be eliminated bymicromolar concentrations of inhibitors.

Many of the active inhibitors are drugs which have already beenadministered as medicines in humans to treat other disorders (notdiabetes).

Example 3 First Preclinical Study

The purpose of this example was to demonstrate how the clinicalusefulness of candidate fructosamine oxidase inhibitors may be assessedusing a standard animal model of diabetes mellitus, thestreptozocin-diabetic rat (STZ rat). This approach took into accountdrug bioavailability, the activity of the drug and its metabolites, andany drug adverse effects or toxicity factors.

Method:

48 Wistar rats aged 6-8 weeks and weighing 200-300 g were randomized:

Group 1 Non-diabetic control

Group 2 Diabetic control

Group 3 Diabetic treated with hydralazine

Group 4 Diabetic treated with EDTA

Group 5 Diabetic treated with hydralazine and acetylcysteine

Group 6 Diabetic treated with acetylcysteine

Streptozotocin (60 mg per kg) was administered into a lateral tail vein.

Non-diabetic controls received a sham injection of buffer. Diabetes wasconfirmed by venous blood glucose measurement >15 mmol/L after 1 weekand diabetic animals were treated with subcutaneous injections ofultralente insulin (4 U/injection) 3-5 days per week to maintain bodygrowth. Medications were administered 50 mg/L in the drinking water overan 8-month period. Timed urine collections and venous plasma sampleswere obtained at monthly intervals.

Results:

Blood glucose control: Rate of conversion to diabetes with intravenousSTZ administration was >95%. Intravenous STZ induced a severe form ofinsulin-dependent diabetes which was sustained over the entire 8-monthduration of the study Despite insulin replacement therapy, glycemiacontrol was poor as evidenced by mean±SD glucose (week 4) and HbA_(1c)(week 32) levels in Table 3.

TABLE 3 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Glucose 9.1 ±1.5 30.1 ± 9.7 35.7 ± 9.5  39.0 ± 6.04 30.4 ± 8.8  37.8 ± 5.2  (mmol/LHbA_(1c)(%) 3.82 ± 0.11 10.85 ± 0.05 8.65 ± 1.18 9.30 ± 0.63 8.72 ± 0.559.47 ± 1.23

(b) Survival: Mortality rate amongst untreated STZ rats was extremelyhigh. Survival was improved significantly by the administration offructosamine oxidase inhibitors (Table 4).

TABLE 4 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Survivors 8 2 65 8 7 at week 32 Signifi- — — ns ns P < 0.025 P < 0.05 cance**Chi-square test compared with untreated STZ rats (Group 2)

The survival curve for STZ rats compared with non-diabetic controls isshown in FIG. 4. Death was presumed secondary to a cardiovascular event.In general, renal function remained normal.

(c) Weight gain: There was a progressive weight gain amongstnon-diabetic controls over the 32 weeks of the study which was abolishedin the STZ diabetic animals. At the end of the 32 week study period,mean weight change amongst surviving study animals was: Group 1, +74.6%;Group 2, −21.0%, Group 3, −11.0%; Group 4, +1.2%, Group 5, +16.0%; andGroup 6, −8.1% (FIG. 5). Compared with untreated diabetic controls,fructosamine oxidase inhibitors caused an improvement in weight gainroughly in proportion to the activity of the inhibitor (Table 2), i.e.,acetylcysteine/hydralazine>EDTA>acetylcysteine>hydralazine.

(d) Clinical Pharmacokinetics:

Hydralazine

The bioavailability of hydralazine in man after oral administration is26-55%. However, only 2.0-3.6% of the drug is excreted in the urineunchanged over 24 hours after oral administration. Most of the drug isrecovered as an inactive acetylated product. See, Talseth T., Eur J ClinPharmacol 10: 395-401 (1976) and Talseth T., Clin Pharmacol Ther 21:715-20 (1977). This could account for the reduced efficacy ofhydralazine as a fructosamine oxidase inhibitor in the current study.Furthermore, drug doses administered to each STZ rat were calculated as12.5 mg hydralazine/day or 35 mg/kg, based on an average consumption of250 mL of water per day and assuming a mean body mass of 350 g. This ratdose far exceeds the maximum recommended human dose of 200 mghydralazine per day (3 mg/kg assuming a mean body mass of 70 kg).

EDTA

The bioavailability of EDTA after oral administration is very low (lessthan 5%) because of poor absorption from the gut limiting its usefulnessin humans to parenteral administration or irrigation techniques. See,for example, Wynn J. E. et al. Toxicol Appl Pharmacol 16: 807-17 (1970).

Acetylcysteine

Acetylcysteine is rapidly absorbed from the gut with a bioavailabilityin man varying between 6 and 10%. See, for example, Borgstrom L. et al.Eur J Clin Pharmacol 31: 217-22 (1986). However, the drug is rapidlydegraded in the liver by elimination of the acetyl moiety. See, forexample, Holdiness M. R., Clin Pharmacokinet 20: 123-34 (1991).Induction of liver enzymes could account for the progressive loss ofdrug efficacy seen after week 12 in the current study.

Conclusions:

Streptozocin induces a severe form of Type I diabetes in the rat with ahigh morbidity and mortality.

Survival of STZ rats was enhanced by treating with fructosamine oxidaseinhibitors in proportion to their activity in an in vitro assay.

Weight gain of STZ rats was enhanced by treating with fructosamineoxidase inhibitors.

There was some benefit in co-administering acetylcysteine andhydralazine suggesting a synergy effect between classes of fructosamineoxidase inhibitors.

Based on these in vivo studies in the rat, the efficacy of a candidatefructosamine oxidase inhibitor, for example, in a human is likely to beinfluenced by the bioavailability of the drug, degradation of the activecompound in vivo, and the maximum oral tolerated dose of the drug.

Example 4 Second Preclinical Study

The purpose of this example was to demonstrate how the clinicalusefulness of candidate fructosamine oxidase inhibitors, alone and incombination, may be assessed using a standard animal model of diabetesmellitus, the streptozocin-diabetic rat (STZ rat). This approach tookinto account drug bioavailability, the activity of the drugs and theirmetabolites, interactions between drugs, and any drug adverse effects ortoxicity factors.

Method:

80 Wistar rats weighing 200-300 g and aged of 6-8 weeks were randomized:

Group 1 Non-diabetic control

Group 2 Diabetic control

Group 3 Diabetic treated with captopril (substrate analogue)

Group 4 Diabetic treated with carbidopa (hydrazine)

Group 5 Diabetic treated with triethylenetetramine dihydrochloride(copper chelator)

Group 6 Diabetic treated with captopril and triethylenetetraminedihydrochloride

Group 7 Diabetic treated with captopril and carbidopa

Group 8 Diabetic treated with triethylenetetramine dihydrochloride andcarbidopa.

Diabetes was induced by administering streptozotocin (60 mg per kg) byintraperitoneal injection. Non-diabetic controls received a shaminjection of buffer. Diabetes was confirmed by venous blood glucosemeasurement >15 mmol/L after 1 week and diabetic animals were treatedwith subcutaneous injections of ultralente insulin (4 U/injection) 3days per week to maintain body growth. Medications were administered ata concentration of 50 mg/L in the drinking water over a 6-month period.Timed urine collections and venous plasma samples were obtained atmonthly intervals. Animals were monitored for blood glucose control andsurvival rate over the course of study. Animals were sacrificed andsubjected to post-mortem examination at the end of the study todetermine various parameters of fructosamine oxidase activity inhibitorefficacy. Parameters include, but are not limited to, survival rate,weight gain, cataract formation, and cardiomyopathy.

Results:

Blood glucose control: Rate of conversion to diabetes withintraperitoneal STZ administration was approximately 80%. Poor glycemiccontrol was sustained over the 6 month duration of the study asevidenced by mean±SD HbA_(1c) (week 4, 12, and 24) levels (Table 5).

TABLE 5 HbA_(1c) Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7Group 8 Week 4 4.1 ± 0.1 8.3 ± 0.1 8.5 ± 0.9 9.0 ± 1.0 8.0 ± 5.2 9.0 ±5.2 9.1 ± 1.5 9.1 ± 1.5 Week 12 4.1 ± 0.1 9.2 ± 0.6 9.2 ± 1.1 9.6 ± 0.78.8 ± 0.9 9.5 ± 0.8 9.5 ± 1.0 9.3 ± 0.9 Week 24 3.7 ± 0.1  9.4 ± 01.3 9.4 ± 01.3 9.9 ± 1.1 9.0 ± 1.4 9.8 ± 1.2 9.8 ± 1.2 9.1 ± 1.2

(b) Survival: Compared with intravenous administration of STZ,intraperitoneal administration of STZ induced a less severe form ofdiabetes with lesser mortality rate. At the end of the 24 week studyperiod, mortality rate amongst study animals was: Group 1, 0%; Group 2,14.3%, Group 3, 0%; Group 4, 0%, Group 5, 0%; Group 6, 12.5%, Group 7,0%, and Group 6, 0%. There was no significant difference between groupsbecause of the low frequency of events.

(c) Weight gain: STZ diabetes caused a profound weight loss in diabeticrats compared with non-diabetic controls. Mean weight gain of studyanimals from the beginning to the end of the 24 week period areindicated in Table 6.

TABLE 6 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8Mean ± SEM 342.8 ± 13.7 54.4 ± 12.5 60.7 ± 20.7 33.7 ± 20.4 123.6 ± 20.556.1 ± 21.3 55.1 ± 17.1 75.8 ± 25.4 weight gain P* — ns ns ns 0.0138 nsns ns *Student's t test compared with untreated STZ rats (Group 2)

Triene administered alone (Group 5) caused a significant improvement onweight gain compared with the untreated STZ diabetic control rat group(Group 2). There was no evidence of synergy between classes offructosamine oxidase inhibitors.

Triethylenetetramine dihydrochloride administered alone (Group 5) causeda significant improvement in weight gain compared with the untreated STZdiabetic control rat group (Group 2). There was no evidence of synergybetween classes of fructosamine oxidase inhibitors.

(d) Cataract formation: Cataract formation has been a recognizedlong-term complication of poorly controlled diabetes. Gross cataractformation in STZ rats compared with diabetic control animals by the endof the study at week 24 is shown in Table 7.

TABLE 7 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8No (%) 0 (0%) 8 (40%) 2 (25%) 2 (25%) 0 (0%) 2 (28%) 5 (62%) 1 (12%)with cataract P* — — ns ns <0.10 ns ns ns *Chi-square test compared withdiabetic control rats (Group 2)

Although not significant at the P=0.05 level, triethylenetetraminedihydrochloride appeared more effective than captopril and carbidopa ininhibiting gross cataract formation. There was no evidence of synergybetween classes of fructosamine oxidase inhibitors.

(e) Diabetic cardiomyopathy: Cardiomyopathy has been a recognizedlong-term complication of poorly controlled diabetes. Macroscopically,hearts of STZ rats were dilated with thinning of the ventricular wall.Sections stained with hematoxylin and eosin and Masson's Trichromeshowed focal pallor with a loss of normal architecture in the myocardiumof both ventricles that began at the sub-endocardial and sub-epicardialregions and spread to encompass the whole ventricular wall in severelyaffected animals. There was also marked infiltration by fibrousconnective tissue between myocytes and increased fibrous connectivetissue in the walls of intramural arteries. These appearances wereconsistent with dilated cardiomyopathy. Gross myocardial fibrosis in STZrats compared with non-diabetic control animals by the end of the studyat week 24 is shown in Table 8.

TABLE 8 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8No (%) 0 10 6 2 0 6 8 7 with severe fibrosis P* — — ns ns <0.005 ns<0.005 ns *Chi-square test compared with diabetic control rats (Group 2)

Triethylenetetramine dihydrochloride was highly effective in inhibitingthe development of diabetic cardiomyopathy. Diabetic cardiomyopathycauses histopathological and functional changes in the heart. Thedisease can be assessed post-mortem by examining the histology.Alternatively, the disease can be assessed ante-mortem by measuringheart function using echocardiography, cardiac catheter studies, ormagnetic resonance imaging of the heart. There was no evidence ofsynergy between classes of fructosamine oxidase inhibitors.

(f) Clinical Pharmacokinetics:

Triethylenetetramine dihydrochloride. The bioavailability oftriethylenetetramine dihydrochloride was less than 10%. Bioavailabilityrefers to the degree to which a drug or other substance becomesavailable to the target tissue after administration. Bioavailability isusually expressed as that proportion of an administered dose that may bemeasured in the blood stream. See, for example, Kodama H. et al., LifeSci 61: 899-907 (1997). Most of the unchanged drug was cleared in theurine within the first 6 hours of oral dosing mainly as an acetylderivative indicating that a three or four times daily drug regimen or asustained release preparation was required. See, for example, Kodama H.et al. supra. In addition, plasma levels in non-fasted rats weresignificantly lower than those observed in fasted animals and the uptakeof triethylenetetramine dihydrochloride from the intestinal brush borderwas competitively inhibited by other amine compounds. See, for example,Tanabe R. et al. J Pharm Pharmacol 48: 517-21 (1996). This implied thattriethylenetetramine dihydrochloride was best administered in thefasting state. Fasting state was usually about half an hour beforemeals. Interference in the absorption of drug from the intestinal brushborder could account for discrepancies between triethylenetetraminedihydrochloride treatment groups (Groups 5, 6, and 8). Finally, in thecurrent study lasting approximately 6 months, each STZ rat consumedapproximately 250 mL of water per day (12.5 mg triethylenetetraminedihydrochloride/rat/day). Assuming a mean body mass of 350 g, this doseof triethylenetetramine dihydrochloride equated to 35 mg/kg. The dose oftriethylenetetramine dihydrochloride previously used in treating humanswith another non-diabetic condition ranged between 1.2-2.4 g (17-35mg/kg assuming a mean body mass 70 kg). See, Walshe J. M. Lancet 8273:643-7 (1982). This implied that humans may be safely treated withcomparable doses of trienes to those administered to rats in the currentstudy to thereby elicit the fructosamine oxidase inhibition and/orantagonism advantages in a diabetic patient referred to herein.

Captopril. The bioavailability of captopril was approximately 65% afteran oral dose. However, the drug was almost completely bound in vivo toalbumin and other plasma proteins and formed inactive mixed disulfideswith endogenous thiols so that plasma levels of active drug might havebeen very low. The elimination half life of unchanged captopril wasapproximately 2 hours. See, Duchin K. L. et al. Clin Pharmacokinet 14:241-59 (1988). These observations might explain the reduced efficacy ofcaptopril in the STZ rat compared with in vitro studies. Furthermore,each STZ rat consumed approximately 12.5 mg captopril/day which equatedto 35 mg/kg assuming a mean body mass of 350 g. This dose far exceededthe maximum recommended human dose of 150 mg captopril per day (2 mg/kgassuming a mean body mass of 70 kg).

Carbidopa. In a study of beagle dogs, the oral absorption of carbidopawas almost complete and the absolute bioavailability was 88%. Thebiological half-life was 5 hours. See, for example, Obach R. et al. JPharm Pharmacol 36: 415-6 (1984). However, carbidopa was an unstablecompound and it degraded naturally in a short period. Solutions left tostand exposed to light at room temperature will undergo 50% oxidativedegradation in 24 hours. See, for example, Pappert E. J. et al. MovementDisorders 12: 608-23 (1997). Reduced bioavailability due to oxidativedegradation of the active drug both prior to its consumption andpost-ingestion in the rat could explain (in part) the reduced efficacyof carbidopa in the current study. Finally, each STZ rat consumedapproximately 12.5 mg carbidopa/day which equated to 35 mg/kg assuming amean body mass of 350 g. This dose far exceeded the maximum recommendedhuman dose of 200 mg carbidopa per day (3 mg/kg assuming a mean bodymass of 70 kg).

Conclusions:

Intraperitoneal streptozocin was associated with a lower mortality ratethan intravenous streptozocin in the rat.

Weight gain over a 6-month period was enhanced in STZ rats treated withthe copper chelator triethylenetetramine dihydrochloride, as shown inTable 6. Captopril and carbidopa were ineffective.

Cataract development may be inhibited by a triene. Efficacy oftriethylenetetramine dihydrochloride was diminished when the drug wasco-administered with either captopril or carbidopa.

The development of diabetic cardiomyopathy was prevented by treatmentwith a triene. Triethylenetetramine dihydrochloride was administered inthe amount of 50 mg of drug per liter in the drinking water. This amountresulted in an average dose of 12.5 mg triethylenetetraminedihydrochloride per rat per day based on an estimated water intake of250 mL per day. In rat groups 6 and 8, triethylenetetraminedihydrochloride was mixed with captopril and carbidopa, respectively, inthe drinking water. Concentrations of triethylenetetraminedihydrochloride, captopril, and carbidopa were all 50 mg of drug perliter of drinking water.

Efficacy of triethylenetetramine dihydrochloride was diminished when thedrug was co-administered with either captopril or carbidopa (50 mg ofdrug per liter of drinking water).

Oral doses of a triene, triethylenetetramine dihydrochloride, whichinhibited the development of complications in the rat (cataract,cardiomyopathy, and early death) were equivalent on a body mass basis todoses of triethylenetetramine dihydrochloride which had previously beenused to treat human beings with another condition (not diabetes).

When administered to humans on a three or four times daily basis or as asustained release preparation in previously tolerated doses of 1.2-2.4g/day, triethylenetetramine dihydrochloride will provide an effectivemeans of treating the long-term complications of diabetes mellitus.

Example 5 Double-Blind, Placebo-Controlled Clinical Trial

The purpose of this example is to demonstrate how the clinicalusefulness of candidate fructosamine oxidase inhibitors is to beassessed in diabetic human subjects. A detailed protocol based on thisproposal has been approved by the Auckland Regional Ethics CommitteeThis approach takes into account drug bioavailability, the activity ofthe drugs and their metabolites, interactions between drugs, any drugadverse effects or toxicity factors and the “scale-up” factor from ratto human treatment.

Objective: This is a pilot study to determine whether a triene reducesthe rate of progression of renal disease and associated microvascularcomplications in patients with diabetic nephropathy due to Type IIdiabetes mellitus.

Patient population: 60 men and women aged between 40 years and 70 yearsof age with poor blood glucose control and diabetic nephropathy due toType II diabetes mellitus. Poor blood glucose control was defined as ahemoglobin A_(1c) (HbA_(1c)) greater than 7% in this group of patientswith advanced microvascular complications, i.e., diabetic nephropathy.Diabetic nephropathy is a clinical syndrome defined as the patienthaving: (i) albuminuria greater than 300 mg per liter; (ii) plasmacreatinine greater than 150 μmol per liter, and (iii) some evidence ofdiabetic retinopathy.

Study design and duration: Randomized double-blind, placebo-controlledstudy design consisting of five periods:

screening period (detecting possible candidates who meet studycriteria);

enrolment period (securing informed consent and baseline measurements);

run-in period (trial of acceptability of study protocols and studymedication);

maintenance period (treatment with drug/placebo, monitoringefficacy/safety);

follow-up period (detect any untoward effect when medication isdiscontinued).

Blinded therapy (a triene 400 mg or placebo) is administered three timesdaily ½ hour before meals in addition to current anti-hypertensive andhypoglycemic therapies. The study terminates when all patients arerandomized and have been in the study (maintenance period) for a minimumof 6 months. All randomized patients who discontinue study drug for anyreason other than death are followed for the entire duration of thestudy; patients who undergo renal transplantation or dialysis arefollowed for vital status only.

Outcomes Efficacy:

The primary outcome measure consists of the rate of decline in renalfunction as measured by glomerular filtration rate (creatinineclearance). Creatinine clearance is a standard means of measuring renalfunction (glomerular filtration rate) in human subjects and since thisprocedure is a standard method and routine to a person of skill in thearts, no further definition or explanation is necessary.

The secondary outcome measures to be evaluated are development ofdiabetic retinopathy, diabetic peripheral neuropathy, and diabeticautonomic neuropathy.

Safety:

Safety parameters evaluated are adverse events and clinical laboratoryabnormalities. Adverse events can be categorized as serious (i.e.,life-threatening) or non-serious. Non-serious adverse events are anyevents, which the clinical investigator may consider to be secondary tothe administration of the drug. Non-limiting examples include headache,nausea, cough, diarrhea, weight loss, alopecia, and impotence. Clinicallaboratory abnormalities are assessed at time points by medical history,physical examination, and laboratory analyses and compared betweengroups. Non-limiting examples include anemia, thrombocytopenia,leukopenia, iron deficiency, disordered liver function tests, andimpaired renal function tests.

Statistical considerations: The sample size estimate for this trial isdetermined for the primary hypothesis that the projected rate of decayof creatinine clearance (1 mL/min) in Type II diabetes mellitus patientswith diabetic nephropathy (creatinine clearance <90 mL/min) is reducedby treating with a triene. The study is powered to detect (80%) a 6mL/min change in creatinine clearance over 6 months with four 2-monthlyreadings (i.e., 0, 2, 4, and 6 months) assuming a 10% rate of loss tofollow-up at the 5% significance level.

CONCLUSIONS

The efficacy of a triene as a treatment of microvascular complicationsin patients with Type II diabetes mellitus is confirmed.

The safety of long-term administration of a triene in patients with poorblood glucose control and diabetic nephropathy due to Type II diabetesmellitus is confirmed.

It also provides a means to determine the clinical usefulness ofalternative fructosamine oxidase inhibitors such as the copper chelatingcompounds D-penicillamine, sar, and diamsar (i.e., triene could be usedin place of placebo in ensuing clinical trials).

1. A method of treating a human for diabetes, the method comprisingadministering to said human a composition comprising a therapeuticallyeffective amount of a pharmaceutically acceptable cupric binding agentselected from the group consisting of triethylenetetraminedihydrochloride, penicillamine, sar, diamsar, and o-phenanthroline. 2.The method of claim 1 wherein said human has type 1 diabetes.
 3. Themethod of claim 1 wherein said human has type 2 diabetes.
 4. The methodof any of claims 1, 2 or 3, wherein said cupric binding agent is acopper chelator.
 5. The method of claim 4, wherein said copper chelatoris administered orally.
 6. A method of treating a human for diabetes,the method comprising administering to said human a compositioncomprising a therapeutically effective amount of a pharmaceuticallyacceptable oxidized copper binding agent.
 7. The method of claim 1wherein said human has type 1 diabetes.
 8. The method of claim 1 whereinsaid human has type 2 diabetes.
 9. The method of any of claims 6, 7, or8, wherein said copper binding agent is a copper chelator.
 10. Themethod of claim 9, wherein said copper chelator is administered orally.11. A method of treating one or more long-term complications of diabetesin a human suffering therefrom, the method comprising administering acomposition consisting essentially of a cupric binding agent in anamount effective to ameliorate one or more of said conditions.
 12. Themethod of claim 11 wherein said human has type 1 diabetes.
 13. Themethod of claim 11 wherein said human has type 2 diabetes.
 14. Themethod of any of claims 11, 12 or 13, wherein said cupric binding agentis a copper chelator.
 15. The method of claim 14, wherein said copperchelator is administered orally.
 16. A method for minimizing or reducingtissue damage associated with diabetes mellitus in a human, whichcomprises the prophylactic administration to a human of atherapeutically effective amount of a cupric binding agent.
 17. Themethod of claim 16 wherein said human has type 1 diabetes.
 18. Themethod of claim 16 wherein said human has type 2 diabetes.
 19. Themethod of any of claims 16, 17 or 18, wherein said cupric binding agentis a copper chelator.
 20. The method of claim 19, wherein said copperchelator is administered orally.
 21. A method of treating one or morelong-term complications of diabetes in a human suffering therefrom, themethod comprising administering a composition consisting essentially ofa compound that can bind or chelate oxidized, copper in an amounteffective to ameliorate one or more of said conditions.
 22. The methodof claim 21 wherein said human has type 1 diabetes.
 23. The method ofclaim 21 wherein said human has type 2 diabetes.
 24. The method of anyof claims 21, 22, or 23, wherein said compound is a copper chelator. 25.The method of claim 21, wherein said copper chelator is administeredorally.
 26. A method for minimizing or reducing tissue damage associatedwith diabetes mellitus in a human, which comprises the prophylacticadministration to a human of a therapeutically effective amount of acompound that can bind or chelate oxidized copper.
 27. The method ofclaim 26 wherein said human has type 1 diabetes.
 28. The method of claim26 wherein said human has type 2 diabetes.
 29. The method of any ofclaims 26, 27, 28, wherein said compound is a copper chelator.
 30. Themethod of claim 29, wherein said copper chelator is administered orally.31. A method of treating one or more long-term complications of diabetesin a human suffering therefrom, the method comprising forming a cupriccomplex within the human by administering a composition consistingessentially of an effective amount of a copper binding agent.
 32. Themethod of claim 31 wherein said human has type 1 diabetes.
 33. Themethod of claim 31 wherein said human has type 2 diabetes.
 34. Themethod of any of claims 31, 32, or 33, wherein said copper binding agentis a copper chelator.
 35. The method according to claim 34 wherein saidcopper chelator is administered orally.
 36. A method of treating one ormore long-term complications of diabetes in a human suffering therefrom,the method comprising forming an oxidized copper complex within thehuman by administering a composition consisting essentially of aneffective amount of a copper binding agent.
 37. The method of claim 36wherein said human has type 1 diabetes.
 38. The method of claim 36wherein said human has type 2 diabetes.
 39. The method of any of claims36, 37, or 38, wherein said copper binding agent is a copper chelator.40. The method according to claim 34 wherein said copper chelator isadministered orally.
 41. A method for minimizing or reducing tissuedamage associated with diabetes mellitus in a human, the methodcomprising forming a cupric complex within the human by prophylacticallyadministering an effective amount of a copper binding agent.
 42. Themethod of claim 41 wherein said human has type 1 diabetes.
 43. Themethod of claim 41 wherein said human has type 2 diabetes.
 44. Themethod of any of claims 41, 42, or 43, wherein said copper binding agentis a copper chelator.
 45. The method according to claim 44 wherein saidcopper chelator is administered orally.
 46. A method for minimizing orreducing tissue damage associated with diabetes mellitus in a human, themethod comprising forming an oxidized copper complex within the human byprophylactically administering an effective amount of a copper bindingagent.
 47. The method of claim 46 wherein said human has type 1diabetes.
 48. The method of claim 46 wherein said human has type 2diabetes.
 49. The method of any of claims 46, 47, or 48, wherein saidcopper binding agent is a copper chelator.
 50. The method according toclaim 49 wherein said copper chelator is administered orally.